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An Automated ELISA to Determine Interleukin-8 Protein Expression in a Human Lung Carcinoma Cell Line

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SAMI® Workstation EX Software was used to develop a Biomek® Assay Workstation protocol for the automation of an ELISA application. The Biomek Assay Workstation1 to 6-96 well Immunotech IL-8 ELISA plates. The IL-8 was induced at 2.5, 5, and 22 hours using varying concentrations of H₂O₂ from the A549 cell line. System used consists of a dual-bridge hybrid Biomek FX, connected to a Cytomat 6000 (Kendro) via a conveyor ALP. An ELx405* (Bio-Tek) and a DTX 880 Multimode Detector are integrated on the deck (Figure 1).

The IL-8 ELISA assay from Immunotech™ was used in this example. H₂O₂ was used to induce an immune response in a human lung carcinoma cell line, A549. The induced immune response in the A549 cells was monitored by the measure of IL-8 protein levels for each of the different levels of H₂O₂ induction. Details of the method and system are discussed below.

System: Hybrid FX

Figure 1. Biomek FX Assay Workstation shown here contains an integrated ELx405 washer, DTX 880 Multimode Detector and Cytomat 6000 incubator linked by a conveyor ALP.

Hardware Setup: Cytomat 6000

The Cytomat 6000 was configured to have 6 ELISA plates in stack 1, 6 P250 tip boxes in stack 2, and 6 CostarDeep96Square deepwells in stack 8.

Hardware Setup: Deck

The deck was configured to have all reagents (4), reagent tip boxes (4), and standards (1) on deck prior to starting the method. The ELx405 was used to wash all plates and holds the wash buffer.

SAMI EX Software Method:

Click image to enlarge

A method was generated using the SAMI EX Workstation software to process up to six (6) 96-well ELISA plates. All plate washing and reading was done on deck as directed by the SAMI EX software. No user intervention was required once the method has started.

Legend: (1) Standards setup and transfer to ELISA plate (standards are diluted in the Biomek method), (2) Sample transfer to ELISA Plate (test samples are in columns 3-12), (3) 2-hour incubation at home, (4) ELx405 wash, (5) Addition of biotinylated anti-IL-8 antibody, (6) Addition of Streptavidin-HRP conjugate, (7) 45-minute incubation at home, (8) ELx405 wash, (9) Addition of Substrate, (10) 30-minute incubation at home, (11) Addition of stop solution, (12) Plates read on the DTX 880 at 450 nm.

Data: Reproducibility Experiment

H₂0₂	Plate 1	Plate 3	Plate 5	H₂0₂	Plate 2	Plate 4	Plate 6
Conc.	2.5 Hour	5 Hour	22 Hour	Conc.	2.5 Hour	5 Hour	22 Hour
	Stim.	Stim.	Stim.		Stim.	Stim.	Stim.
0	0.09%	4.36%	1.86%	0	8.07%	1.18%	3.20%
100	0.10%	8.67%	4.63%	600	4.41%	1.39%	7.27%
200	2.04%	6.01%	2.06%	700	2.50%	3.55%	4.53%
300	3.55%	8.67%	4.68%	800	0.51%	4.93%	6.87%
400	2.06%	7.44%	4.42%	900	1.43%	6.72%	6.88%
500	5.68%	8.85%	5.40%	1000	1.12%	4.28%	1.46%
Average	2.25%	7.34%	3.84%	Average	3.01%	3.68%	5.04%

To confirm that the IL-8 protein expression levels are consistent when using the automated ELISA method, six different concentrations of H₂O₂ were used to induce an immune response in the A549 cells. C.V.s were calculated and compared for two different runs. As shown in the table below, consistent results were obtained in the two runs with an average C.V. of less than 8%.

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For Research Use Only; not for use in diagnostic procedures.

For comments or questions about T³ Update, please contact David Daniels, Ph.D., editor.